Project Summary The prevalence of post-traumatic stress disorder (PTSD) is nearly 2:1 in women compared with men, yet the neurobiological basis of this sex difference is unknown. This project addresses this gap, in accordance with NIMH Strategic Objective 1, by determining how the stress neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) functionally modulates fear circuitry and complex fear behavior, differentially in females and males. The central hypothesis driving this work is informed by clinical data linking this peptide system with PTSD. Women, but not men, with a genetic polymorphism within the gene encoding the type 1 receptor for PACAP (PAC1R) exhibit maladaptive responding to threat-related stimuli: heightened reactivity to threatening cues and deficits in the ability to learn to discriminate fear and safe cues. Recent work by the PI showed that interfering with PAC1R signaling within the prefrontal cortex impairs the formation of cued fear memory in female but not male rats, using a variant of fear conditioning called trace fear conditioning, which requires sustained attention to fear cues and depends on working-memory like neuronal activity within the prefrontal cortex. Females also had higher levels of PAC1R compared with males in that study. The objective of this proposal is to determine how PACAP-PAC1R signaling modulates activity within the fear circuitry needed for learning about threat and safety: the prefrontal cortex and its connection with the amygdala. The approach uses multiple levels of analysis to elucidate the basic biology linking sex-driven changes in peptidergic signaling and physiological changes in prefrontal-amygdala circuits to alterations in complex fear behavior (NIMH Strategy 1.1, Research Priority D). The central hypothesis is that PACAP-PAC1R signaling in the prefrontal cortex is dynamically regulated by estradiol and promotes both the strength and accuracy of fear memories in females, in part by increasing the excitability and synaptic regulation of amygdala-projecting prefrontal neurons. In Aim 1, the capacity for PACAP-PAC1R signaling to modulate intrinsic excitability and synaptic regulation of prefrontal cells connected with the basolateral amygdala will be assessed using ex vivo electrophysiology. In Aim 2, the ability of PACAP to enhance fear memories yet protect against fear generalization will be tested with local manipulation of PACAP signaling in the prefrontal cortex during trace conditioning and fear discrimination learning. In Aim 3, the capacity for estradiol to drive the expression of PAC1R and the consequence on memory of knocking down PAC1R locally in the prefrontal cortex using shRNA will be examined. Importantly, males and females will be included in all experiments to determine how PACAP contributes to the sexual dimorphism of the prefrontal cortex and the degree to which PACAP has the capacity to influence fear learning and behavior in males. The proposed work is significant because it is expected to advance our current understanding of sexual dimorphism in the prefrontal cortex as well as to provide a neurobiological substrate for maladaptive fear learning observed in women with PTSD.